The invention relates to the use of T cell tyrosine phosphatase (TC-PTP) to modulate cellular sensitivity to DNA damaging agents, and to regulate cell cycle.
Protein phosphorylation is a common regulatory mechanism used by cells to selectively modify proteins carrying regulatory signals from outside the cell to the nucleus. The proteins that execute these biochemical modifications are a group of enzymes known as protein kinases and protein phosphatases. The first protein tyrosine phosphatase was characterized over a decade ago by Tonks et al. (1988) J. Biol. Chem. 263:6722-6730, and since then a great number of other family members have been cloned and biochemically characterized. Yet, the biological function is known for only a few family members.
One of the earliest reported PTP enzymes was the T-cell protein tyrosine phosphatase (TC-PTP). The cDNA encoding the TC-PTP was originally isolated from a human T-cell library (Cool et al. (1989) Proc. Natl. Acad. Sci. 86:5257-5261), although it is widely expressed. There are highly related homologues in mouse and rat, under the respective name of MPTP and PTPS (Mosinger et al. (1992) P.N.A.S. 89:499-503; Radha et al. (1997) FEBS Lett 409:33-36).
Although TC-PTP was one of the first phosphatases identified, the function(s) of this PTP is unknown. A potential role for TC-PTP in receptor kinase signaling was proposed based on the specific association of the epidermal growth factor receptor (EGFR) and the SHC adaptor protein to the substrate trapping TC-PTP C216S mutant (Tiganis et al. (1998) Mol. Cell. Biol. 18:1622-1634). Another aspect of TC-PTP function is suggested by reports that TC-PTP mRNA levels fluctuate in a cell cycle specific manner. TC-PTP mRNA levels appear to increase in G0 and early G1, and decrease for the rest of the cell cycle (Tillmann et al., supra.) On the contrary, the protein levels of the rat PTPS homologue do not appear to vary during the cell cycle, but seemingly changes between nuclear and cytoplasmic compartments. A similar variance in localization was also reported for the human 45 kDa protein.
In a recent publication of the TC-PTP knock-out mouse (You-Ten et al. (1997) J. Exp. Med. 186:683-693), it was found that homozygous animals.die between 3-5 weeks of age, in part because of severe anemia due to a failure of erythropoiesis. The TC-PTPxe2x88x92/xe2x88x92 mice have a defective microenvironment of the bone marrow resulting from a near absence of stromal cells, and an inability of T and B cells to proliferate following general cell activation by either Concanavalin A or lipopolysaccharides (LPS).
The cell cycle is regulated by a complex network of interacting proteins whose activity is modulated by phosphorylation reactions. This regulation provides a coordinated downstream process leading to DNA: replication. The cell cycle is mainly controlled by two different protein families: the cyclin-dependent kinases (Cdks) and their regulatory subunits, cyclins, Sherr et al. (1996) Science 274:1672-1677. The assembly and disassembly of specific cyclin/Cdk complexes are pivotal events driving the cell cycle. Progression through the G1 phase is controlled by two different complexes: cyclin D/Cdk4,6 which is active in early G1, and cyclin E/Cdk2 which is highly active in late G1. The main substrate of both complexes is the product of the retinoblastoma gene, Rb. Rb protein is known as a repressor of the progression toward S phase. Once Rb is phosphorylated in early G1 by cyclin D/Cdk4,6 and by the cyclin E/Cdk2 in late G1, its affinity for E2F transcription factor decreases and initiates transcription of important genes for the S phase.
In the multistep progression of cancer, a normal cell may lose or gain several regulatory cues, thereby leading to its metamorphosis into unregulated proliferation. Included in these changes are signaling events that influence the cell cycle, DNA repair, mitotic and apoptotic properties of the oncogenic cells. In view of the importance of DNA repair and cell cycle regulation in both normal development and the tumorigenic process, the signaling events, mechanism(s) of action, and modulation provided by and placed on TC-PTP are of great interest.
Methods and compositions are provided for modulating cellular sensitivity to DNA damaging agents through manipulation of T cell protein tyrosine phosphatase (TC-PTP) activity. Also provided are methods of regulating the progression through cell cycle by altering TC-PTP activity. The phenotypic characterization of cells lacking TC-PTP demonstrates a defective progression through the cell cycle, and sensitivity to DNA damaging agents. Screening assays are provided for selecting agents that affect the activity of TC-PTP, including assays relating to the interaction of TC-PTP with its substrate, p62dok.
In one embodiment of the invention, inhibitors of TC-PTP activity are used to induce, sensitivity to DNA damaging agents, e.g. to sensitize susceptible tumors to DNA damaging chemo- or radiation therapy. Inhibitors include dominant negative mutants, inhibitory fragments or mutants of TC-PTP substrate proteins, anti-sense nucleic acids, small molecule inhibitors, and the like.
In another embodiment of the invention, TC-PTP activity is upregulated or otherwise provided to a cell as a protection against DNA damage. Of particular interest is the provision of TC-PTP activity to patients having acute or chronic sensitivity to DNA damage, e.g. ataxia telangiectasia, and other diseases having a defect in DNA repair.